Highly sensitive and specific detection of human papillomavirus type 16 using CRISPR/Cas12a assay coupled with an enhanced single nanoparticle dark-field microscopy imaging technique.

Human papillomavirus (HPV) is a prevalent sexually transmitted pathogen associated with cervical cancer. Detecting high-risk HPV (hr-HPV) infections is crucial for cervical cancer prevention, particularly in resource-limited settings. Here, we present a highly sensitive and specific sensor for HPV-16 detection based on CRISPR/Cas12a coupled with enhanced single nanoparticle dark-field microscopy (DFM) imaging techniques. Ag-Au satellites were assembled through the hybridization of AgNPs-based spherical nucleic acid (Ag-SNA) and AuNPs-based spherical nucleic acid (Au-SNA), and their disassembly upon target-mediated cleavage by the Cas12a protein was monitored using DFM for HPV-16 quantification. To enhance the cleavage efficiency and detection sensitivity, the composition of the ssDNA sequences on Ag-SNA and Au-SNA was optimized. Additionally, we explored using the SynSed technique (synergistic sedimentation of Brownian motion suppression and dehydration transfer) as an alternative particle transfer method in DFM imaging to traditional electrostatic deposition. This addresses the issue of inconsistent deposition efficiency of Ag-Au satellites and their disassembly due to their size and charge differences. The sensor achieved a remarkable limit of detection (LOD) of 10 fM, lowered by 9-fold compared to traditional electrostatic deposition methods. Clinical testing in DNA extractions from 10 human cervical swabs demonstrated significant response differences between the positive and negative samples. Our sensor offers a promising solution for sensitive and specific HPV-16 detection, with implications for cancer screening and management.

Homogeneous immunoassay utilizing fluorescence resonance energy transfer from quantum dots to tyramide dyes deposited on full immunocomplexes.

There is an urgent need for homogeneous immunoassays that offer sufficient sensitivity for routine clinical practice. In this study, we have developed a highly sensitive, fluorescence resonance energy transfer (FRET)-based homogeneous immunoassay. Unlike previous FRET-based homogeneous immunoassays, where acceptors were attached to antibody molecules located far from the donor, we employed acceptors to label the entire sandwich-structured immunocomplex, including two antibodies and one antigen. As a result, the FRET signal was amplified by a factor of 10, owing to the reduced distance between the donor and acceptors. We validated our method by quantifying carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) in PBS buffer and blank plasma. The limits of detection (LOD) for CEA and AFP in both PBS buffer and blank plasma were comparable, reaching sub-femtomolar levels. Furthermore, we successfully quantified CEA and AFP in three human plasma samples, thereby confirming the reliability of our method for clinical applications.

Corrigendum: Contactless and robust dielectric microspheres-assisted surface-enhanced Raman scattering sensitivity improvement for anthrax biomarker detection.

[This corrects the article DOI: 10.3389/fchem.2022.1057241.].

Contactless and robust dielectric microspheres-assisted surface-enhanced Raman scattering sensitivity improvement for anthrax biomarker detection.

This report presents a contactless and robust dielectric microspheres (DMs)-assisted surface enhanced Raman scattering (SERS) enhancement method to improve SERS detection sensitivity detection sensitivity. DMs that could focus and collect light were embedded within the polydimethylsiloxane (PDMS) film to avoid direct contact with the analytical solution and improve detection reliability. The as prepared DMs embedded PDMS (DMs-PDMS) film was integrated with a microfluidic technique to enhance the SERS signal of a liquid substrate. Detection in microfluidic systems can reduce reagent consumption, shorten assay time, and avoid evaporation of the colloid substrate solution. The robustness and potential influencing factors of DMs-PDMS film assisted SERS enhancement (DERS) were evaluated using 4-aminothiophenol (4-ATP) as the Raman probe. The sensing performance of the proposed method toward dipicolinic acid (DPA) was evaluated, and an evident signal intensification was obtained. Remarkably, the DMs-PDMS film can also be implemented on solid substrates. A proof-of-concept experiment was performed by covering the DMs-PDMS film directly over an AgNPs@Si solid substrate wherein a 5.7-fold sensitivity improvement was achieved.

The Arsenic-Binding Aptamer Cannot Bind Arsenic: Critical Evaluation of Aptamer Selection and Binding.

An arsenic-binding aptamer named Ars-3 was reported in 2009, and it has been used for detection of As(III) in more than two dozen papers. In this work, we performed extensive binding assays using isothermal titration calorimetry, various DNA-staining dyes, and gold nanoparticles. By carefully comparing Ars-3 and a few random control DNA sequences, no specific binding of As(III) was observed in each case. Therefore, we conclude that Ars-3 cannot bind As(III). Possible reasons for some of the previously reported binding and detection were speculated to be related to the adsorption of As(III) onto gold surfaces, which were used in many related sensor designs, and As(III)/Au interactions were not considered before. The selection data in the original paper were then analyzed in terms of sequence alignment, secondary structure prediction, and dissociation constant measurement. These steps need rigorous testing before confirming specific binding of newly selected aptamers. This study calls for attention to the gap between aptamer selection and biosensor design, and the gap needs to be filled by careful binding assays to further the growth of the aptamer field.

Adsorption of Arsenite on Gold Nanoparticles Studied with DNA Oligonucleotide Probes.

Gold nanoparticles (AuNPs) have been extensively used for detecting arsenite, As(III). Many methods rely on a DNA aptamer that claimed to bind specifically to inorganic arsenic. In these cases, the focus was on arsenic binding to the aptamer, while the potential interactions between As(III) and the AuNP surface were ignored. Herein, a set of spectroscopic and isothermal titration calorimetry (ITC) experiments were conducted to measure the adsorption of As(III) by AuNPs and its competition with DNA adsorption. With 10 mM As(III), 18% of adsorbed DNA was displaced from AuNPs, while preadsorption of only 20 µM As(III) inhibited DNA adsorption by around 50%. The affinity of As(III) on AuNPs is comparable to Br- and guanosine. ITC and Raman spectroscopy both indicated that only As(III) can be adsorbed, while As(V) had no measurable interactions with the AuNPs. Based on this understanding, a random DNA sequence was used and a similar colorimetric response in the presence of As(III) was observed. This study confirmed the affinity between As(III) and the gold surface. The As(III)/gold interaction is strong enough to affect DNA adsorption, and care should be taken to interpret the observations based on the color change of AuNPs for the detection of As(III).

In situ synthesis of low-cost and large-scale flexible metal nanoparticle-polymer composite films as highly sensitive SERS substrates for surface trace analysis.

Surface-enhanced Raman spectroscopy (SERS) has been one of the most promising analytical tools. Despite many efforts in the design of SERS substrates, it remains a great challenge for creating a general flexible substrate that could in situ detect analytes on diverse objects. Herein, we report our attempt to address this issue by developing a facile and versatile method capable of generating silver/gold nanoparticles in situ on the surface of a cellulose acetate (CA) polymer in a simple, cheap, practical, and capping agent-free way. The as-prepared substrates exhibit excellent sensitivity, which enabled detection of Rhodamine 6G at concentrations as low as 10-12 M. Taking advantage of the excellent flexibility and optical transparency of the CA matrix, the highly SERS-active substrate was applied for in situ identification and detection of pesticide residues on fruits. The results indicated that tetramethylthiuram disulfide (TMTD) and thiabendazole (TBZ) can be clearly identified at concentrations as low as 18.05 ng cm-2 and 15.1 ng cm-2, respectively, which were much lower than the maximum permitted residue doses with respect to food safety.

A Single-Molecule Homogeneous Immunoassay by Counting Spatially "Overlapping" Two-Color Quantum Dots with Wide-Field Fluorescence Microscopy.

We developed a single-molecule homogeneous immunoassay by counting spatially "overlapping" two-color quantum dots (QD) under a wide-field fluorescence microscope. QD 655 with red fluorescence and QD 565 with green fluorescence were modified with capture and detection antibodies, respectively. A capture antibody-modified QD 655 and a detection antibody-modified QD 565 were conjugated by a corresponding antigen molecule to form a "sandwich" immunocomplex. The conjugated QD 655 could not be distinguished from the conjugated QD 565 by fluorescent microscopy because the distance between them was smaller than the resolution of an optical microscope (approximately 200 nm). The immunocomplex color became yellow because of the spatial "overlap" of the red and green fluorescence. The number of the yellow spots was equal to the number of immunocomplex molecules, while the concentration of the antigen was related to the ratio of the yellow dots to the red dots. The successful quantification of two model proteins in the human plasma, namely, alpha-fetoprotein and carcinoembryonic antigen, demonstrated the accuracy and reliability of our approach.

Visual and colorimetric determination of H2O2 and glucose based on citrate-promoted H2O2 sculpturing of silver nanoparticles.

Isotropic silver nanoparticles (iAg NPs) can be easily prepared at low costs, have a low electrochemical potential and high extinction coefficient. An effective colorimetric assay for H2O2 is reported here based on the finding that H2O2 can induce the shape transformation of citrate-capped iAg NPs with the help of citrate. The substantial shape variation affords an apparent surface plasmon resonance (SPR) shift, accompanied by a vivid color change from light yellow to mauve. The color change can be observed visually if the concentration of H2O2 is 2 µM or higher. A good linear relationship was obtained over the concentration range of 0.2-32 µM with a limit of detection of 90 nM. By making use of glucose oxidase, the method is further extended to glucose detection. Glucose at a concentration as low as 10 µM can be well determined with bare eyes. Benefitting from the high selectivity, the detection of glucose in human serum is realized, and the results are in good agreement with those provided by a clinical analyzer. Graphical abstract Schematic presentation of a colorimetric assay for H2O2 and glucose based on citrate-promoted H2O2-mediated shape transformation of the isotropic silver nanoparticles (Ag NPs). The shape variation of isotropic Ag NPs induces a color change from light yellow to mauve.

Sensing Active Heparin by Counting Aggregated Quantum Dots at Single-Particle Level.

Developing highly sensitive and highly selective assays for monitoring heparin levels in blood is required during and after surgery. In previous studies, electrostatic interactions are exploited to recognize heparin and changes in light signal intensity are used to sense heparin. In the present study, we developed a quantum dot (QD) aggregation-based detection strategy to quantify heparin. When cationic micelles and fluorescence QDs modified with anti-thrombin III (AT III) are added into heparin sample solution, the AT III-QDs, which specifically bind with heparin, aggregate around the micelles. The aggregated QDs are recorded by spectral imaging fluorescence microscopy and differentiated from single QDs based on the asynchronous process of blue shift and photobleaching. The ratio of aggregated QD spots to all counted QD spots is linearly related to the amount of heparin in the range of 4.65 × 10 -4 U/mL to 0.023 U/mL. The limit of detection is 9.3 × 10 -5 U/mL (∼0.1 nM), and the recovery of the spiked heparin at 0.00465 U/mL (∼5 nM) in 0.1% human plasma is acceptable.

Viscosity based droplet size controlling in negative pressure driven droplets generator for large-scale particle synthesis.

The poor control and regulation of droplets limit the applications of negative pressure driven droplet generator (NPDDG). Here, we present a simple method to control droplet size in NPDDG via varying the oil viscosity. Depending on the infinite intersolubility of high viscous mineral oil and low viscous hexadecane, we obtain a series of oils with kinematic viscosities linearly varied from 4.2 to 194.6 mm2 /s. By using these oils as continuous phases, monodisperse droplets are fabricated with controllable size in NPDDG. This viscosity-based droplet regulation method is effective, reliable, and compatible with scale-up processes. Compared with droplet generator driven by positive pressure, the presented method can fabricate hydrogel particles massively, without complicated multilayer chip structure and complex fluid controlling, which may extend the potential of NPDDG in droplets based high-throughput assay or large-scale materials synthesis.

A self-driven miniaturized liquid fuel cell.

We present a miniaturized fuel cell driven by an evaporation pump. The prototype cell shows a net peak current density of 22 mA cm-2 and a net power density of 10.2 mW cm-2, both of which are the highest net values among passive-driven micro-fuel cells.

Fabrication of Janus droplets by evaporation driven liquid-liquid phase separation.

We present a universal and scalable method to fabricate Janus droplets based on evaporation driven liquid-liquid phase separation. In this work, the morphologies and chemical properties of separate parts of the Janus droplets can be flexibly regulated, and more complex Janus droplets (such as core-shell Janus droplets, ternary Janus droplets, and multiple Janus droplets) can be constructed easily.

Single Gold Nanoparticle-Based Colorimetric Detection of Picomolar Mercury Ion with Dark-Field Microscopy.

Mercury severely damages the environment and human health, particularly when it accumulates in the food chain. Methods for the colorimetric detection of Hg(2+) have increasingly been developed over the past decade because of the progress in nanotechnology. However, the limits of detection (LODs) of these methods are mostly either comparable to or higher than the allowable maximum level (10 nM) in drinking water set by the US Environmental Protection Agency. In this study, we report a single Au nanoparticle (AuNP)-based colorimetric assay for Hg(2+) detection in solution. AuNPs modified with oligonucleotides were fixed on the slide. The fixed AuNPs bound to free AuNPs in the solution in the presence of Hg(2+) because of oligonucleotide hybridization. This process was accompanied by a color change from green to yellow as observed under an optical microscope. The ratio of changed color spots corresponded with Hg(2+) concentration. The LOD was determined as 1.4 pM, which may help guard against mercury accumulation. The proposed approach was applied to environmental samples with recoveries of 98.3 ± 7.7% and 110.0 ± 8.8% for Yuquan River and industrial wastewater, respectively.

Counting quantum dot aggregates for the detection of biotinylated proteins.

We develop a method to quantify the quantum dots (QDs) in QD aggregates in aqueous solution by recording the entire process of blue shifting and photobleaching under continuous illumination and utilize this method to detect the biotinylated proteins based on counting the degree of aggregation (DOA).

In situ formation of phosphorescent molecular gold(I) cluster in a macroporous polymer film to achieve colorimetric cyanide sensing.

A highly phosphorescent molecular Au(I) cluster capable of rapid, sensitive, and selective detection of cyanide has been successfully fabricated. The origin of the outstanding sensing performance of the molecular Au(I) cluster toward cyanide is justified by X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses. The response mechanism employed with the molecular Au(I) cluster and the cost-effectiveness in cyanide detection affords several key sensor features, making this molecular Au(I) cluster-based sensor unique compared to other cyanide sensing schemes. Importantly, by exploring the phosphorescent properties of the molecular Au(I) cluster in solid state, we demonstrate the first example of the molecular gold(I) cluster-based macroporous sensing film for colorimetric detection of cyanide in complex samples, including red wine, coffee, juice, and soil. Remarkably, the as-prepared sensing film inherits the sensing ability of the molecular Au(I) cluster, and offers a high mechanical flexibility and novel opportunities for real-time monitoring cyanide release in cassava manufacturing.

Template-free synthesis of renewable macroporous carbon via yeast cells for high-performance supercapacitor electrode materials.

The urgent need for sustainable development has forced material scientists to explore novel materials for next-generation energy storage devices through a green and facile strategy. In this context, yeast, which is a large group of single cell fungi widely distributed in nature environments, will be an ideal candidate for developing effective electrode materials with fascinating structures for high-performance supercapacitors. With this in mind, herein, we present the first example of creating three-dimensional (3D) interpenetrating macroporous carbon materials via a template-free method, using the green, renewable, and widespread yeast cells as the precursors. Remarkably, when the as-prepared materials are used as the electrode materials for supercapacitors, they exhibit outstanding performance with high specific capacitance of 330 F g(-1) at a current density of 1 A g(-1), and good stability, even after 1000 charge/discharge cycles. The approach developed in this work provides a new view of making full use of sustainable resources endowed by nature, opening the avenue to designing and producing robust materials with great promising applications in high-performance energy-storage devices.

Engineering natural materials as surface-enhanced Raman spectroscopy substrates for in situ molecular sensing.

Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical tool. However, its applications for in situ detection of target molecules presented on diverse material surfaces have been hindered by difficulties in rapid fabricating SERS-active substrates on the surfaces of these materials through a simple, low-cost, and portable approach. Here, we demonstrate our attempt to address this issue by developing a facile and versatile method capable of in situ generating silver nanoparticle film (SNF) on the surfaces of both artificial and natural materials in a simple, cheap, practical, and disposable manner. Taking advantage of the high SERS enhancement ability of the prepared SNF, the proposed strategy can be used for in situ inspecting herbicide and pesticide residues on vegetables, as well as the abuse of antiseptic in aquaculture industry. Therefore, it opens new avenues for advancing the application prospects of SERS technique in the fields of food safety, drug security, as well as environment monitoring.

Fluorescent silver nanoclusters for user-friendly detection of Cu2+ on a paper platform.

The development of a user-friendly sensing platform for the detection of Cu(2+) in water is necessary as there are wide concerns due to the substantial impact of Cu(2+) on human health, environmental monitoring, and so on. Motivated by this, we report a fluorescent silver nanoclusters (AgNCs)-based sensor for the detection of Cu(2+). These water-soluble AgNCs, as a new class of fluorescent probes, were synthesized by using azobenzene modified poly(acrylic acid) (MPAA) as templates. Their fluorescence can be quenched in the presence of Cu(2+), which enables the label-free detection of Cu(2+) in real water samples. Furthermore, such AgNCs can be integrated onto cellulose filter paper and used as fluorescent indicators for Cu(2+). The fluorescence quenching can be observed by the naked eye under UV light. It should be noted that this AgNCs-based paper assay performs successfully in barrelled drinking water and river water samples. Therefore, it opens up new avenues to the development of robust clusters-based sensing platforms.

Dual modal in vivo imaging using upconversion luminescence and enhanced computed tomography properties.

In vivo upconversion luminescence (UCL) imaging, exhibiting favorable characteristics such as high photostability, no blinking, sharp emission lines, and long lifetimes, is recognized as the excellent and significant photoluminescence imaging for the future. To develop the imaging system with high visual sensitivity and tissue penetration, the functional molecules with X-ray computed tomography (CT) contrast were grafted onto upconversion nanoparticles to obtain ß-NaYF(4):18% Yb(3+),2%Er(3+)@SiO(2)-I/PEG (UCNPs@SiO(2)-I/PEG) nanoprobes. These nanoprobes are water-soluble, have low cytotoxicity, and possess excellent UCL and remarkable CT contrast. Of particular note is that, besides the element iodine, rare earth elements (Y, Yb, and Er) present in the nanoprobes also show CT contrast. Moreover, no background autofluorescence signal is found in in vivo UCL images. We believe that these nanoprobes with dual modal in vivo imaging of UCL and CT can serve as a promising platform for clinical diagnosis or biomedical studies.